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Abstract:

The embodiments provide Compound PC-5,
[2-((S)-2-malonylamino-6-amino-hexanoyl amino)-ethyl]-ethyl-carbamic acid
hydromorphone ester, or acceptable salts, solvates, and hydrates thereof.
The present disclosure also provides pharmaceutical compositions, and
their methods of use, where the pharmaceutical compositions comprise a
prodrug, Compound PC-5, that provides enzymatically-controlled release of
hydromorphone, and, optionally, a trypsin inhibitor that interacts with
the enzyme that mediates the enzymatically-controlled release of
hydromorphone from the prodrug so as to attenuate enzymatic cleavage of
the prodrug.

7. The composition of claim 6, wherein the trypsin inhibitor is an
arginine mimic or a lysine mimic.

8. The composition of claim 7, wherein the arginine mimic or lysine mimic
is a synthetic compound.

9. The composition of claim 6, wherein the trypsin inhibitor is a
compound of formula: ##STR00047## wherein: Q1 is selected from
--O-Q4 or -Q4-COOH, where Q4 is C1-C4 alkyl;
Q2 is N or CH; and Q3 is aryl or substituted aryl.

10. The composition of claim 6, wherein the trypsin inhibitor is a
compound of formula: ##STR00048## wherein: Q5 is --C(O)--COOH or
--NH-Q6-Q7-SO2--C6H5, where Q6 is
--(CH2)p--COOH; Q7 is --(CH2)r--C6H5;
Q8 is NH; n is a number from zero to two; o is zero or one; p is an
integer from one to three; and r is an integer from one to three.

11. The composition of claim 6, wherein the trypsin inhibitor is a
compound of formula: ##STR00049## wherein: Q5 is --C(O)--COOH or
--NH-Q6-Q7-SO2--C6H5, where Q6 is
--(CH2)p--COOH; Q7 is --(CH2)r--C6H5;
and p is an integer from one to three; and r is an integer from one to
three.

13. The composition of claim 6, wherein the trypsin inhibitor is a
compound of formula: ##STR00051## wherein X is NH; n is zero or one;
Lt1 is selected from --C(O)--O--; --O--C(O)--;
--O--(CH2)m--O--; --OCH2--Art2-CH2O--;
--C(O)--NRt3--; and --NRt3--C(O)--; Rt3 is selected from
hydrogen, C1-6 alkyl, and substituted C1-6 alkyl; Art1 and
Art2 are independently a substituted or unsubstituted aryl group; m
is a number from 1 to 3; and Rt2 is selected from hydrogen, halogen,
nitro, alkyl, substituted alkyl, alkoxy, carboxyl, alkoxycarbonyl, acyl,
aminoacyl, guanidine, amidino, carbamide, amino, substituted amino,
hydroxyl, cyano and
--(CH2)m--C(O)--O--(CH2)m--C(O)--N--Rn1Rn2,
wherein each m is independently zero to 2; and Rn1 and Rn2 are
independently selected from hydrogen and C1-4 alkyl.

14. The composition of claim 6, wherein the trypsin inhibitor is a
compound of formula: ##STR00052## wherein each X is NH; each n is
independently zero or one; Lt1 is selected from --C(O)--O--;
--O--C(O)--; --O--(CH2)m--O--;
--OCH2--Art2-CH2O--; --C(O)--NRt3--; and
--NRt3--C(O)--; Rt3 is selected from hydrogen, C1-6 alkyl,
and substituted C1-6 alkyl; Art1 and Art2 are
independently a substituted or unsubstituted aryl group; and m is a
number from 1 to 3.

17. A method of treating or preventing pain in a patient in need thereof,
which comprises administering an effective amount of a composition of
claim 6 to the patient.

18. (canceled)

19. A method for reducing drug abuse potential of a composition
containing a compound of claim 1, the method comprising: combining
Compound PC-5 with a trypsin inhibitor, wherein the trypsin inhibitor
reduces the ability of a user to release hydromorphone from Compound PC-5
by addition of trypsin.

20. A composition comprising: a prodrug comprising hydromorphone
covalently bound to a promoiety comprising a trypsin-cleavable moiety,
wherein cleavage of the trypsin-cleavable moiety by trypsin mediates
release of hydromorphone, wherein the prodrug is Compound PC-5, shown
below: ##STR00053## or salt, solvate, or hydrate thereof; and a trypsin
inhibitor that interacts with the trypsin that mediates
enzymatically-controlled release of hydromorphone from the prodrug
following ingestion of the composition.

21. A dose unit comprising the composition of claim 20, wherein the
prodrug and trypsin inhibitor are present in the dose unit in an amount
effective to provide for a pre-selected pharmacokinetic (PK) profile
following ingestion.

22. The dose unit of claim 21, wherein the pre-selected PK profile
comprises at least one PK parameter value that is less than the PK
parameter value of hydromorphone released following ingestion of an
equivalent dosage of the prodrug in the absence of inhibitor.

23. The dose unit of claim 22, wherein the PK parameter value is selected
from a hydromorphone Cmaxvalue, a hydromorphone exposure value, and a
(1/hydromorphone Tmax) value.

24. The dose unit of claim 21, wherein the dose unit provides for a
pre-selected PK profile following ingestion of at least two dose units.

25. The dose unit of claim 24, wherein the pre-selected PK profile is
modified relative to the PK profile following ingestion of an equivalent
dosage of the prodrug in the absence of inhibitor.

26. The dose unit of claim 24, wherein the dose unit provides that
ingestion of an increasing number of the dose units provides for a linear
PK profile.

27. The dose unit of claim 24, wherein the dose unit provides that
ingestion of an increasing number of the dose units provides for a
nonlinear PK profile.

28. The dose unit of claim 24, wherein the PK parameter value is selected
from a hydromorphone Cmaxvalue, a (1/hydromorphone Tmax) value, and a
hydromorphone exposure value.

29. A composition comprising: a container suitable for containing a
composition for administration to a patient; and a dose unit comprising
the composition of claim 20 disposed within the container.

30. The composition of claim 20, wherein the composition is a dose unit
having a total weight of from 1 microgram to 2 grams.

31. The composition of claim 20, wherein the composition has a combined
weight of prodrug and trypsin inhibitor of from 0.1% to 99% per gram of
the composition.

32. A method to treat a patient comprising administering a pharmaceutical
composition according to claim 20 to a patient in need thereof.

33. A method of making a dose unit, the method comprising: combining in a
dose unit: a prodrug comprising hydromorphone covalently bound to a
promoiety cleavable by trypsin, wherein cleavage of the promoiety by the
trypsin mediates release of hydromorphone from the prodrug, wherein the
prodrug is Compound PC-5 shown below: ##STR00054## and a trypsin
inhibitor that interacts with the trypsin that mediates
enzymatically-controlled release of hydromorphone from the prodrug;
wherein the prodrug and trypsin inhibitor are present in the dose unit in
an amount effective to attenuate release of hydromorphone from the
prodrug such that ingestion of multiples of dose units by a patient does
not provide a proportional release of hydromorphone.

34. A method of claim 33, wherein said release of drug is decreased
compared to release of drug by an equivalent dosage of prodrug in the
absence of inhibitor.

35. A method for identifying a prodrug and a trypsin inhibitor suitable
for formulation in a dose unit, the method comprising: combining a
prodrug, a trypsin inhibitor, and trypsin in a reaction mixture, wherein
the prodrug comprises hydromorphone covalently bound to a promoiety
comprising a trypsin-cleavable moiety, wherein cleavage of the
trypsin-cleavable moiety by trypsin mediates release of hydromorphone; or
administering to an animal a prodrug and a trypsin inhibitor, wherein the
prodrug comprises hydromorphone covalently bound to a promoiety
comprising a trypsin-cleavable moiety, wherein cleavage of the
trypsin-cleavable moiety by trypsin mediates release of hydromorphone; or
administering to an animal tissue a prodrug and a trypsin inhibitor,
wherein the prodrug comprises hydromorphone covalently bound to a
promoiety comprising a trypsin-cleavable moiety, wherein cleavage of the
trypsin-cleavable moiety by trypsin mediates release of hydromorphone,
wherein the prodrug is Compound PC-5, shown below: ##STR00055## and
detecting prodrug conversion, wherein a decrease in prodrug conversion in
the presence of the trypsin inhibitor as compared to prodrug conversion
in the absence of the trypsin inhibitor indicates the prodrug and trypsin
inhibitor are suitable for formulation in a dose unit.

36. (canceled)

37. The method of claim 35, wherein said administering comprises
administering to the animal increasing doses of inhibitor co-dosed with a
selected fixed dose of prodrug.

38. The method of claim 35, wherein said detecting facilitates
identification of a dose of inhibitor and a dose of prodrug that provides
for a pre-selected pharmacokinetic (PK) profile.

39. The method of claim 35, wherein said method comprises an in vivo
assay.

Description:

INTRODUCTION

[0001] Phenolic opioids are susceptible to misuse, abuse, or overdose. Use
of and access to these drugs therefore needs to be controlled. The
control of access to the drugs is expensive to administer and can result
in denial of treatment for patients that are not able to present
themselves for dosing. For example, patients suffering from acute pain
may be denied treatment with an opioid unless they have been admitted to
a hospital. Furthermore, control of use is often ineffective, leading to
substantial morbidity and deleterious social consequences.

[0006] The present disclosure also provides a prodrug comprising
hydromorphone covalently bound to a promoiety comprising a
trypsin-cleavable moiety, wherein cleavage of the trypsin-cleavable
moiety by trypsin mediates release of hydromorphone, wherein the prodrug
is Compound PC-5 and an optional trypsin inhibitor.

[0007] The present disclosure also provides pharmaceutical compositions,
and their methods of use, where the pharmaceutical compositions comprise
a prodrug, Compound PC-5, that provides enzymatically-controlled release
of hydromorphone, and, optionally, a trypsin inhibitor that interacts
with the enzyme that mediates the enzymatically-controlled release of
hydromorphone from the prodrug so as to attenuate enzymatic cleavage of
the prodrug. The disclosure provides for the enzyme being trypsin.

BRIEF DESCRIPTION OF THE FIGURES

[0008]FIG. 1 is a schematic representing the effect of increasing the
level of a trypsin inhibitor ("inhibitor", X axis) on a PK parameter
(e.g., drug Cmax) (Y axis) for a fixed dose of prodrug. The effect of
inhibitor upon a prodrug PK parameter can range from undetectable, to
moderate, to complete inhibition (i.e., no detectable drug release).

[0009]FIG. 2 provides schematics of drug concentration in plasma (Y axis)
over time. Panel A is a schematic of a pharmacokinetic (PK) profile
following ingestion of prodrug with a trypsin inhibitor (dashed line)
where the drug Cmaxis modified relative to that of prodrug without
inhibitor (solid line). Panel B is a schematic of a PK profile following
ingestion of prodrug with inhibitor (dashed line) where drug Cmaxand drug
Tmax are modified relative to that of prodrug without inhibitor (solid
line). Panel C is a schematic of a PK profile following ingestion of
prodrug with inhibitor (dashed line) where drug Tmax is modified relative
to that of prodrug without inhibitor (solid line).

[0010]FIG. 3 provides schematics representing differential
concentration-dose PK profiles that can result from the dosing of
multiples of a dose unit (X axis) of the present disclosure. Different PK
profiles (as exemplified herein for a representative PK parameter, drug
Cmax (Y axis)) can be provided by adjusting the relative amount of
prodrug and trypsin inhibitor contained in a single dose unit or by using
a different prodrug or inhibitor in the dose unit.

[0011]FIG. 4A and FIG. 4B compare mean plasma concentrations over time of
hydromorphone release following PO administration of increasing doses of
prodrug Compound PC-5 to rats.

[0013]FIG. 6A and FIG. 6B compare mean plasma concentrations over time of
hydromorphone release following PO administration of a single dose unit
and of multiple dose units of a composition comprising prodrug Compound
PC-5 and trypsin inhibitor Compound 109 to rats.

[0014]FIG. 7 compares mean plasma concentrations over time of prodrug
Compound PC-5 and hydromorphone following IV administration of prodrug
Compound PC-5 to rats.

[0015]FIG. 8 demonstrates release of hydromorphone from prodrug Compound
PC-5 exposed in vitro to a variety of household chemicals and enzyme
preparations.

DEFINITIONS

[0016] The following terms have the following meaning unless otherwise
indicated. Any undefined terms have their art recognized meanings.

[0017] "Dose unit" as used herein refers to a combination of a
trypsin-cleavable prodrug (e.g., trypsin-cleavable prodrug) and a trypsin
inhibitor. A "single dose unit" is a single unit of a combination of a
trypsin-cleavable prodrug (e.g., trypsin-cleavable prodrug) and a trypsin
inhibitor, where the single dose unit provide a therapeutically effective
amount of drug (i.e., a sufficient amount of drug to effect a therapeutic
effect, e.g., a dose within the respective drug's therapeutic window, or
therapeutic range). "Multiple dose units" or "multiples of a dose unit"
or a "multiple of a dose unit" refers to at least two single dose units.

[0018] "PK profile" refers to a profile of drug concentration in blood or
plasma. Such a profile can be a relationship of drug concentration over
time (i.e., a "concentration-time PK profile") or a relationship of drug
concentration versus number of doses ingested (i.e., a
"concentration-dose PK profile"). A PK profile is characterized by PK
parameters.

[0019] "PK parameter" refers to a measure of drug concentration in blood
or plasma, such as: 1) "drug Cmax", the maximum concentration of drug
achieved in blood or plasma; 2) "drug Tmax", the time elapsed following
ingestion to achieve Cmax; and 3) "drug exposure", the total
concentration of drug present in blood or plasma over a selected period
of time, which can be measured using the area under the curve (AUC) of a
time course of drug release over a selected period of time (t).
Modification of one or more PK parameters provides for a modified PK
profile.

[0020] "Pharmacodynamic (PD) profile" refers to a profile of the efficacy
of a drug in a patient (or subject or user), which is characterized by PD
parameters. "PD parameters" include "drug Emax" (the maximum drug
efficacy), "drug EC50" (the concentration of drug at 50% of the Emax) and
side effects.

[0021] "Gastrointestinal enzyme" or "GI enzyme" refers to an enzyme
located in the gastrointestinal (GI) tract, which encompasses the
anatomical sites from mouth to anus. Trypsin is an example of a GI
enzyme.

[0022] "Gastrointestinal enzyme-cleavable moiety" or "GI enzyme-cleavable
moiety" refers to a group comprising a site susceptible to cleavage by a
GI enzyme. For example, a "trypsin-cleavable moiety" refers to a group
comprising a site susceptible to cleavage by trypsin.

[0023] "Gastrointestinal enzyme inhibitor" or "GI enzyme inhibitor" refers
to any agent capable of inhibiting the action of a gastrointestinal
enzyme on a substrate. The term also encompasses salts of
gastrointestinal enzyme inhibitors. For example, a "trypsin inhibitor"
refers to any agent capable of inhibiting the action of trypsin on a
substrate.

[0024] "Pharmaceutical composition" refers to at least one compound and
can further comprise a pharmaceutically acceptable carrier, with which
the compound is administered to a patient.

[0026] The term "solvate" as used herein refers to a complex or aggregate
formed by one or more molecules of a solute, e.g. a prodrug or a
pharmaceutically-acceptable salt thereof, and one or more molecules of a
solvent. Such solvates are typically crystalline solids having a
substantially fixed molar ratio of solute and solvent. Representative
solvents include by way of example, water, methanol, ethanol,
isopropanol, acetic acid, and the like. When the solvent is water, the
solvate formed is a hydrate.

[0027] "Pharmaceutically acceptable carrier" refers to a diluent,
adjuvant, excipient or vehicle with, or in which a compound is
administered.

[0028] "Patient" includes humans, and also other mammals, such as
livestock, zoo animals and companion animals, such as a cat, dog or
horse.

[0029] "Preventing" or "prevention" or "prophylaxis" refers to a reduction
in risk of occurrence of a condition, such as pain.

[0030] "Prodrug" refers to a derivative of an active agent that requires a
transformation within the body to release the active agent. In certain
embodiments, the transformation is an enzymatic transformation. Prodrugs
are frequently, although not necessarily, pharmacologically inactive
until converted to the active agent.

[0031] "Promoiety" refers to a form of protecting group that, when used to
mask a functional group within an active agent, converts the active agent
into a prodrug. Typically, the promoiety will be attached to the drug via
bond(s) that are cleaved by enzymatic or non-enzymatic means in vivo.

[0032] "Treating" or "treatment" of any condition, such as pain, refers,
in certain embodiments, to ameliorating the condition (i.e., arresting or
reducing the development of the condition). In certain embodiments
"treating" or "treatment" refers to ameliorating at least one physical
parameter, which may not be discernible by the patient. In certain
embodiments, "treating" or "treatment" refers to inhibiting the
condition, either physically, (e.g., stabilization of a discernible
symptom), physiologically, (e.g., stabilization of a physical parameter),
or both. In certain embodiments, "treating" or "treatment" refers to
delaying the onset of the condition.

[0033] "Therapeutically effective amount" means the amount of a compound
(e.g., prodrug) that, when administered to a patient for preventing or
treating a condition such as pain, is sufficient to effect such
treatment. The "therapeutically effective amount" will vary depending on
the compound, the condition and its severity and the age, weight, etc.,
of the patient.

DETAILED DESCRIPTION

[0034] Before the present invention is further described, it is to be
understood that this invention is not limited to particular embodiments
described, as such may, of course, vary. It is also to be understood that
the terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting, since the scope of
the present invention will be limited only by the appended claims.

[0035] It must be noted that as used herein and in the appended claims,
the singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. It is further noted that the
claims may be drafted to exclude any optional element. As such, this
statement is intended to serve as antecedent basis for use of such
exclusive terminology as "solely," "only" and the like in connection with
the recitation of claim elements, or use of a "negative" limitation.

[0036] It should be understood that as used herein, the term "a" entity or
"an" entity refers to one or more of that entity. For example, a compound
refers to one or more compounds. As such, the terms "a", "an", "one or
more" and "at least one" can be used interchangeably. Similarly the terms
"comprising", "including" and "having" can be used interchangeably.

[0037] The publications discussed herein are provided solely for their
disclosure prior to the filing date of the present application. Nothing
herein is to be construed as an admission that the present invention is
not entitled to antedate such publication by virtue of prior invention.
Further, the dates of publication provided may be different from the
actual publication dates which may need to be independently confirmed.

[0038] Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although any methods
and materials similar or equivalent to those described herein can also be
used in the practice or testing of the present invention, the preferred
methods and materials are now described. All publications mentioned
herein are incorporated herein by reference to disclose and describe the
methods and/or materials in connection with which the publications are
cited.

[0039] Except as otherwise noted, the methods and techniques of the
present embodiments are generally performed according to conventional
methods well known in the art and as described in various general and
more specific references that are cited and discussed throughout the
present specification. See, e.g., Loudon, Organic Chemistry, Fourth
Edition, New York: Oxford University Press, 2002, pp. 360-361, 1084-1085;
Smith and March, March's Advanced Organic Chemistry: Reactions,
Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001.

[0040] The nomenclature used herein to name the subject compounds is
illustrated in the Examples herein. When possible, this nomenclature has
generally been derived using the commercially-available AutoNom software
(MDL, San Leandro, Calif.).

[0041] It is appreciated that certain features of the invention, which
are, for clarity, described in the context of separate embodiments, may
also be provided in combination in a single embodiment. Conversely,
various features of the invention, which are, for brevity, described in
the context of a single embodiment, may also be provided separately or in
any suitable sub-combination. All combinations of the embodiments
pertaining to the chemical groups represented by the variables are
specifically embraced by the present invention and are disclosed herein
just as if each and every combination was individually and explicitly
disclosed, to the extent that such combinations embrace compounds that
are stable compounds (i.e., compounds that can be isolated,
characterised, and tested for biological activity). In addition, all
sub-combinations of the chemical groups listed in the embodiments
describing such variables are also specifically embraced by the present
invention and are disclosed herein just as if each and every such
sub-combination of chemical groups was individually and explicitly
disclosed herein.

[0043] Compounds as described herein can be purified by any of the means
known in the art, including chromatographic means, such as high
performance liquid chromatography (HPLC), preparative thin layer
chromatography, flash column chromatography and ion exchange
chromatography. Any suitable stationary phase can be used, including
normal and reversed phases as well as ionic resins. See, e.g.,
Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R.
Snyder and J. J. Kirkland, John Wiley and Sons, 1979; and Thin Layer
Chromatography, ed E. Stahl, Springer-Verlag, New York, 1969.

[0044] During any of the processes for preparation of the compounds of the
present disclosure, it may be necessary and/or desirable to protect
sensitive or reactive groups on any of the molecules concerned. This can
be achieved by means of conventional protecting groups as described in
standard works, such as T. W. Greene and P. G. M. Wuts, "Protective
Groups in Organic Synthesis", Fourth edition, Wiley, New York 2006. The
protecting groups can be removed at a convenient subsequent stage using
methods known from the art.

[0045] The compounds described herein can contain one or more chiral
centers and/or double bonds and therefore, can exist as stereoisomers,
such as double-bond isomers (i.e., geometric isomers), enantiomers or
diastereomers. Accordingly, all possible enantiomers and stereoisomers of
the compounds including the stereoisomerically pure form (e.g.,
geometrically pure, enantiomerically pure or diastereomerically pure) and
enantiomeric and stereoisomeric mixtures are included in the description
of the compounds herein. Enantiomeric and stereoisomeric mixtures can be
resolved into their component enantiomers or stereoisomers using
separation techniques or chiral synthesis techniques well known to the
skilled artisan. The compounds can also exist in several tautomeric forms
including the enol form, the keto form and mixtures thereof. Accordingly,
the chemical structures depicted herein encompass all possible tautomeric
forms of the illustrated compounds.

[0046] The compounds described also include isotopically labeled compounds
where one or more atoms have an atomic mass different from the atomic
mass conventionally found in nature. Examples of isotopes that can be
incorporated into the compounds disclosed herein include, but are not
limited to, 2H, 3H, 11C, 13C, 14C, 5N,
18O, 17O, etc. Compounds can exist in unsolvated forms as well
as solvated forms, including hydrated forms. In general, compounds can be
hydrated or solvated. Certain compounds can exist in multiple crystalline
or amorphous forms. In general, all physical forms are equivalent for the
uses contemplated herein and are intended to be within the scope of the
present disclosure.

Representative Embodiments

[0047] Reference will now be made in detail to various embodiments. It
will be understood that the invention is not limited to these
embodiments. To the contrary, it is intended to cover alternatives,
modifications, and equivalents as may be included within the spirit and
scope of the allowed claims.

[0052] The disclosure provides Compound PC-5, a phenol-modified
hydromorphone prodrug which provides enzymatically-controlled release of
hydromorphone. In Compound PC-5, a promoiety is attached to hydromorphone
via modification of the phenol moiety in which the hydrogen atom of the
phenolic hydroxyl group of hydromorphone is replaced by a covalent bond
to the promoiety.

[0053] In Compound PC-5, the promoiety comprises a cyclizable spacer
leaving group and a cleavable moiety. In Compound PC-5, the
phenol-modified hydromorphone prodrug is a corresponding compound in
which the phenolic hydrogen atom has been substituted with a spacer
leaving group bearing a nitrogen nucleophile that is protected with an
enzymatically-cleavable moiety, the configuration of the spacer leaving
group and nitrogen nucleophile being such that, upon enzymatic cleavage
of the cleavable moiety, the nitrogen nucleophile is capable of forming a
cyclic urea, liberating the compound from the spacer leaving group so as
to provide hydromorphone.

[0054] The enzyme capable of cleaving the enzymatically-cleavable moiety
may be a peptidase, also referred to as a protease--the promoiety
comprising the enzymatically-cleavable moiety being linked to the
nucleophilic nitrogen through an amide (e.g. a peptide: --NHC(O)--) bond.
In some embodiments, the enzyme is a digestive enzyme of a protein. The
disclosure provides for the enzyme being trypsin and for the
enzymatically-cleavable moiety being a trypsin-cleavable moiety.

[0055] The corresponding prodrug provides post administration-activated,
controlled release of hydromorphone. The prodrug requires enzymatic
cleavage to initiate release of hydromorphone and thus the rate of
release of hydromorphone depends upon both the rate of enzymatic cleavage
and the rate of cyclization. Accordingly, the prodrug has reduced
susceptibility to accidental overdosing or abuse, whether by deliberate
overdosing, administration through an inappropriate route, such as by
injection, or by chemical modification using readily available household
chemicals. The prodrug is configured so that it will not provide
excessively high plasma levels of the active drug if it is administered
inappropriately, and cannot readily be decomposed to afford the active
drug other than by enzymatic cleavage followed by controlled cyclization.

[0056] The cyclic group formed when hydromorphone is released is
conveniently pharmaceutically acceptable, in particular a
pharmaceutically acceptable cyclic urea. It will be appreciated that
cyclic ureas are generally very stable and have low toxicity.

General Synthetic Procedures for Compound PC-5

[0057] Compound PC-5 can be synthesized using the methods described in WO
2007/140272. Compound PC-5 may be obtained via the routes generically
illustrated in Scheme 1.

[0059] Referring now to Scheme 1, where for illustrative purposes T is NH,
Y is N(CH2CH3), W is NH, p is one, R4 is a side chain of
lysine, and R5 is --C(O)CH2C(O)OH, X is a phenolic opioid, P is
a protecting group, and M is a leaving group, compound PC1-1 may be
acylated with an appropriate carboxylic acid or carboxylic acid
equivalent to provide compound PC1-2 which then may be deprotected to
yield compound PC1-3. Compound PC1-3 is then reacted with an activated
carbonic acid equivalent PC1-4 to provide compound PC1-5. Compound PC-5
may be obtained via the routes generically illustrated in Scheme 2.

##STR00006## ##STR00007##

[0060] In Scheme 2, a solution of N-ethylethylenediamine and
trifluoroacetate is refluxed in a suitable solvent, such as acetonitrile
and water, to form Compound S-A. Then, a carboxybenzyl group (Cbz group
or Z group) is attached to Compound S-A to form Compound S-B. Methods of
protecting an amino group with Cbz group are known in the art and include
use of reagents, such as N-(benzyloxycarbonyl)succinimide or
benzylchloroformate. Then, Compound S-B is subjected to conditions to
remove the trifluoroacetate group to form Compound S-C. Suitable
conditions to remove the trifluoroacetate group include hydrolysis, such
as use of lithium hydroxide.

[0061] With further reference to Scheme 2, Compound S-C is coupled with
Fmoc-Lys(Boc)-OH to form Compound S-D. Standard peptide coupling reagents
can be used for the reaction. Suitable peptide coupling reagents include,
but are not limited to, EDCI and HOBt, Pybrop and diisopropylethylamine,
or HATU. Then, the Fmoc group is removed from Compound S-D to give
Compound S-E. Suitable conditions to remove the Fmoc group include basic
conditions, such as use of piperidine.

[0062] Then, a malonyl group is attached to Compound S-E via a reaction
with mono-tert-butyl malonate. Reaction between Compound S-E and
mono-tert-butyl malonate can be aided with use of activation reagents,
such as symmetric anhydrides,
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
(HBTU), dicyclohexylcarbodiimide (DCC) diisopropylcarbodiimide
(DIC)/1-hydroxybenzotriazole (HOBt), and
benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate
(BOP). Then, the Cbz group is removed from Compound S-F to give Compound
S-G. Suitable conditions to remove the Cbz group include hydrogenation.

[0063] With further reference to Scheme 2, Compound S-G is coupled with
protected hydromorphone to give Compound S-H. Hydromorphone is protected
at the phenol group as a carbonate by a reaction between hydromorphone
hydrochloride and 4-nitrophenyl chloroformate. Compound S-G and the
protected hydromorphone are coupled to form Compound S-H. To couple
Compound S-G and the protected hydromorphone, suitable activating
reagents that aid in the coupling reaction can be used. Suitable
activating agents include triazolols, such as hydroxybenzotriazole (HOBt)
and 1-hydroxy-7-aza-benzotriazole (HOAt), and carbodiimides, such as
dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC).

[0064] Finally, the Boc group and tert-butyl group of Compound S-H are
removed to yield Compound PC-5. The Boc group and tert-butyl group can be
removed with acidic conditions. Suitable reagents that can be used for
the deprotection reaction include trifluoroacetic acid and hydrochloric
acid.

Trypsin Inhibitors

[0065] As disclosed herein, the present disclosure also provides
pharmaceutical compositions, and their methods of use, where the
pharmaceutical compositions comprise a prodrug, Compound PC-5, that
provides enzymatically-controlled release of hydromorphone, and a trypsin
inhibitor that interacts with the enzyme that mediates the
enzymatically-controlled release of hydromorphone from the prodrug so as
to attenuate enzymatic cleavage of the prodrug. The disclosure provides
for the enzyme being trypsin.

[0066] As used herein, the term "trypsin inhibitor" refers to any agent
capable of inhibiting the action of trypsin on a substrate. The term
"trypsin inhibitor" also encompasses salts of trypsin inhibitors. The
ability of an agent to inhibit trypsin can be measured using assays well
known in the art. For example, in a typical assay, one unit corresponds
to the amount of inhibitor that reduces the trypsin activity by one
benzoyl-L-arginine ethyl ester unit (BAEE-U). One BAEE-U is the amount of
enzyme that increases the absorbance at 253 nm by 0.001 per minute at pH
7.6 and 25° C. See, for example, K. Ozawa, M. Laskowski, 1966, J.
Biol. Chem. 241, 3955 and Y. Birk, 1976, Meth. Enzymol. 45, 700. In
certain instances, a trypsin inhibitor can interact with an active site
of trypsin, such as the 51 pocket and the S3/4 pocket. The 51 pocket has
an aspartate residue which has affinity for a positively charged moiety.
The S3/4 pocket is a hydrophobic pocket. The disclosure provides for
specific trypsin inhibitors and non-specific serine protease inhibitors.

[0067] There are many trypsin inhibitors known in the art, both those
specific to trypsin and those that inhibit trypsin and other proteases
such as chymotrypsin. The disclosure provides for trypsin inhibitors that
are proteins, peptides, and small molecules. The disclosure provides for
trypsin inhibitors that are irreversible inhibitors or reversible
inhibitors. The disclosure provides for trypsin inhibitors that are
competitive inhibitors, non-competitive inhibitors, or uncompetitive
inhibitors. The disclosure provides for natural, synthetic or
semi-synthetic trypsin inhibitors.

[0068] Trypsin inhibitors can be derived from a variety of animal or
vegetable sources: for example, soybean, corn, lima and other beans,
squash, sunflower, bovine and other animal pancreas and lung, chicken and
turkey egg white, soy-based infant formula, and mammalian blood. Trypsin
inhibitors can also be of microbial origin: for example, antipain; see,
for example, H. Umezawa, 1976, Meth. Enzymol. 45, 678. A trypsin
inhibitor can also be an arginine or lysine mimic or other synthetic
compound: for example arylguanidine, benzamidine,
3,4-dichloroisocoumarin, diisopropylfluorophosphate, gabexate mesylate,
phenylmethanesulfonyl fluoride, or substituted versions or analogs
thereof. In certain embodiments, trypsin inhibitors comprise a covalently
modifiable group, such as a chloroketone moiety, an aldehyde moiety, or
an epoxide moiety. Other examples of trypsin inhibitors are aprotinin,
camostat and pentamidine.

[0069] As used herein, an arginine or lysine mimic is a compound that is
capable of binding to the P1 pocket of trypsin and/or interfering
with trypsin active site function. The arginine or lysine mimic can be a
cleavable or non-cleavable moiety.

[0070] In one embodiment, the trypsin inhibitor is derived from soybean.
Trypsin inhibitors derived from soybean (Glycine max) are readily
available and are considered to be safe for human consumption. They
include, but are not limited to, SBTI, which inhibits trypsin, and
Bowman-Birk inhibitor, which inhibits trypsin and chymotrypsin. Such
trypsin inhibitors are available, for example from Sigma-Aldrich, St.
Louis, Mo., USA.

[0071] It will be appreciated that the pharmaceutical composition
according to the embodiments may further comprise one or more other
trypsin inhibitors.

[0072] As stated above, a trypsin inhibitor can be an arginine or lysine
mimic or other synthetic compound. In certain embodiments, the trypsin
inhibitor is an arginine mimic or a lysine mimic, wherein the arginine
mimic or lysine mimic is a synthetic compound.

[0073] Certain trypsin inhibitors include compounds of formula:

##STR00008##

[0074] wherein:

[0075] Q1 is selected from --O-Q4 or -Q4-COOH, where
Q4 is C1-C4 alkyl;

[0114] Rt14 represents a hydrogen atom, a C1-4 alkyl group
substituted by a phenyl group or a group of formula: COORt17,
wherein Rt17 represents a hydrogen atom, a C1-4 alkyl group or
a C1-4 alkyl group substituted by a phenyl group;

[0115] provided that Rt11, Rt12 and Rt13 do not represent
simultaneously hydrogen atoms;

[0116] or nontoxic salts, acid addition salts or hydrates thereof.

[0117] In certain embodiments, the trypsin inhibitor is a compound
selected from the following:

##STR00025##

[0118] In certain embodiments, the trypsin inhibitor is a compound of
formula T-II:

[0146] It is to be appreciated that the invention also includes inhibitors
of other enzymes involved in protein assimilation that can be used in
combination with a prodrug disclosed herein comprising an amino acid of
alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,
glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or
valine or amino acid variants thereof. An amino acid variant refers to an
amino acid that is modified from a naturally-occurring amino acid but
still comprises activity similar to that of the naturally-occurring amino
acid.

Combinations of Prodrug and Trypsin Inhibitor

[0147] As discussed above, the present disclosure provides pharmaceutical
compositions which comprise a trypsin inhibitor and Compound PC-5, a
phenol-modified hydromorphone prodrug, that comprises a promoiety
comprising a trypsin-cleavable moiety that, when cleaved, facilitates
release of phenolic opioid. Examples of compositions containing Compound
PC-5 and a trypsin inhibitor are described below.

[0148] The embodiments provide a pharmaceutical composition, which
comprises a compound of Formulae T-I to T-IV and Compound PC-5, or a
pharmaceutically acceptable salt thereof.

[0149] The embodiments provide a pharmaceutical composition, which
comprises Compound 109 and Compound PC-5, or a pharmaceutically
acceptable salt thereof.

[0150] Certain embodiments provide for a combination of Compound PC-5 and
a trypsin inhibitor, in which the trypsin inhibitor is shown in the
following table.

[0151] The disclosure provides for Compound PC-5 and a further prodrug or
drug included in a pharmaceutical composition. Such a prodrug or drug
would provide additional analgesia or other benefits. Examples include
opioids, acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs)
and other analgesics. In one embodiment, Compound PC-5 would be combined
with an opioid antagonist prodrug or drug. Other examples include drugs
or prodrugs that have benefits other than, or in addition to, analgesia.
The embodiments provide a pharmaceutical composition, which comprises
Compound PC-5 and acetaminophen, or a pharmaceutically acceptable salt
thereof.

[0152] Such compositions can also comprise a trypsin inhibitor. In certain
embodiments, the trypsin inhibitor is selected from SBTI, BBSI, Compound
101, Compound 106, Compound 108, Compound 109, and Compound 110. In
certain embodiments, the trypsin inhibitor is Compound 109. In certain
embodiments, the trypsin inhibitor is camostat.

[0153] In certain embodiments, a pharmaceutical composition can comprise
Compound PC-5, a non-opioid drug and at least one opioid or opioid
prodrug.

Pharmaceutical Compositions and Methods of Use

[0154] As disclosed herein, the embodiments provide a composition, which
comprises Compound PC-5, or [2-((S)-2-malonylamino-6-amino-hexanoyl
amino)-ethyl]-ethyl-carbamic acid hydromorphone ester. The pharmaceutical
composition according to the embodiments can further comprise a
pharmaceutically acceptable carrier. The composition is conveniently
formulated in a form suitable for oral (including buccal and sublingual)
administration, for example as a tablet, capsule, thin film, powder,
suspension, solution, syrup, dispersion or emulsion. The composition can
contain components conventional in pharmaceutical preparations, e.g. one
or more carriers, binders, lubricants, excipients (e.g., to impart
controlled release characteristics), pH modifiers, sweeteners, bulking
agents, coloring agents or further active agents.

[0155] Patients can be humans, and also other mammals, such as livestock,
zoo animals and companion animals, such as a cat, dog or horse.

[0156] In another aspect, the embodiments provide a pharmaceutical
composition as described hereinabove for use in the treatment of pain.
The pharmaceutical composition according to the embodiments is useful,
for example, in the treatment of a patient suffering from, or at risk of
suffering from, pain. Accordingly, the present disclosure provides
methods of treating or preventing pain in a subject, the methods
involving administering to the subject a disclosed composition. The
present disclosure provides for a disclosed composition for use in
therapy or prevention or as a medicament. The present disclosure also
provides the use of a disclosed composition for the manufacture of a
medicament, especially for the manufacture of a medicament for the
treatment or prevention of pain.

[0158] The present disclosure also provides use of Compound PC-5 in the
treatment of pain. The present disclosure also provides use of Compound
PC-5 in the prevention of pain.

[0159] The present disclosure provides use of Compound PC-5 in the
manufacture of a medicament for treatment of pain. The present disclosure
provides use of Compound PC-5 in the manufacture of a medicament for
prevention of pain.

[0160] In another aspect, the embodiments provide a method of treating
pain in a patient requiring treatment, which comprises administering an
effective amount of a pharmaceutical composition as described
hereinabove. In another aspect, the embodiments provides method of
preventing pain in a patient requiring treatment, which comprises
administering an effective amount of a pharmaceutical composition as
described hereinabove.

[0161] The amount of composition disclosed herein to be administered to a
patient to be effective (i.e. to provide blood levels of hydromorphone
sufficient to be effective in the treatment or prophylaxis of pain) will
depend upon the bioavailability of the particular composition, the
susceptibility of the particular composition to enzyme activation in the
gut, as well as other factors, such as the species, age, weight, sex, and
condition of the patient, manner of administration and judgment of the
prescribing physician. If the composition also comprises a trypsin
inhibitor, the amount of composition disclosed herein to be administered
to a patient would also depend on the amount and potency of trypsin
inhibitor present in the composition. In general, the composition dose
can be such that Compound PC-5 is in the range of from 0.01 milligrams
prodrug per kilogram to 20 milligrams prodrug per kilogram (mg/kg) body
weight. For example, a composition comprising Compound PC-5 can be
administered at a dose equivalent to administering free hydromorphone in
the range of from 0.02 to 0.5 mg/kg body weight or 0.01 mg/kg to 10 mg/kg
body weight or 0.01 to 2 mg/kg body weight. In one embodiment, the
composition can be administered at a dose such that the level of
hydromorphone achieved in the blood is in the range of from 0.5 ng/ml to
10 ng/ml.

[0162] As disclosed above, the present disclosure also provides
pharmaceutical compositions which comprise a trypsin inhibitor and
Compound PC-5, a phenol-modified hydromorphone prodrug, that comprises a
promoiety comprising a trypsin-cleavable moiety that, when cleaved,
facilitates release of hydromorphone. In such pharmaceutical
compositions, the amount of a trypsin inhibitor to be administered to the
patient to be effective (i.e. to attenuate release of hydromorphone when
administration of Compound PC-5 alone would lead to overexposure of
hydromorphone) will depend upon the effective dose of Compound PC-5 and
the potency of the particular trypsin inhibitor, as well as other
factors, such as the species, age, weight, sex and condition of the
patient, manner of administration and judgment of the prescribing
physician. In general, the dose of trypsin inhibitor can be in the range
of from 0.05 mg to 50 mg per mg of Compound PC-5. In a certain
embodiment, the dose of trypsin inhibitor can be in the range of from
0.001 mg to 50 mg per mg of Compound PC-5. In one embodiment, the dose of
trypsin inhibitor can be in the range of from 0.01 nanomoles to 100
micromoles per micromole of Compound PC-5.

[0163] The present disclosure provides dose units of prodrug and inhibitor
that can provide for a desired pharmacokinetic (PK) profile. Dose units
can provide a modified PK profile compared to a reference PK profile as
disclosed herein. It will be appreciated that a modified PK profile can
provide for a modified pharmacodynamic (PD) profile. Ingestion of
multiples of such a dose unit can also provide a desired PK profile.

[0164] Unless specifically stated otherwise, "dose unit" as used herein
refers to a combination of a trypsin-cleavable prodrug and a trypsin
inhibitor. A "single dose unit" is a single unit of a combination of a
trypsin-cleavable prodrug and a trypsin inhibitor, where the single dose
unit provide a therapeutically effective amount of drug (i.e., a
sufficient amount of drug to effect a therapeutic effect, e.g., a dose
within the respective drug's therapeutic window, or therapeutic range).
"Multiple dose units" or "multiples of a dose unit" or a "multiple of a
dose unit" refers to at least two single dose units.

[0165] As used herein, a "PK profile" refers to a profile of drug
concentration in blood or plasma. Such a profile can be a relationship of
drug concentration over time (i.e., a "concentration-time PK profile") or
a relationship of drug concentration versus number of doses ingested
(i.e., a "concentration-dose PK profile".) A PK profile is characterized
by PK parameters.

[0166] As used herein, a "PK parameter" refers to a measure of drug
concentration in blood or plasma, such as: 1) "drug Cmax", the maximum
concentration of drug achieved in blood or plasma; 2) "drug Tmax", the
time elapsed following ingestion to achieve Cmax; and 3) "drug exposure",
the total concentration of drug present in blood or plasma over a
selected period of time, which can be measured using the area under the
curve (AUC) of a time course of drug release over a selected period of
time (t). Modification of one or more PK parameters provides for a
modified PK profile.

[0167] For purposes of describing the features of dose units of the
present disclosure, "PK parameter values" that define a PK profile
include drug Cmax (e.g., hydromorphone Cmax), total drug exposure (e.g.,
area under the curve) (e.g., hydromorphone exposure) and 1/(drug Tmax)
(such that a decreased 1/Tmax is indicative of a delay in Tmax relative
to a reference Tmax) (e.g., 1/hydromorphone Tmax). Thus a decrease in a
PK parameter value relative to a reference PK parameter value can
indicate, for example, a decrease in drug Cmax, a decrease in drug
exposure, and/or a delayed Tmax.

[0168] Dose units of the present disclosure can be adapted to provide for
a modified PK profile, e.g., a PK profile that is different from that
achieved from dosing a given dose of prodrug in the absence of inhibitor
(i.e., without inhibitor). For example, dose units can provide for at
least one of decreased drug Cmax, delayed drug Tmax and/or decreased drug
exposure compared to ingestion of a dose of prodrug in the same amount
but in the absence of inhibitor. Such a modification is due to the
inclusion of an inhibitor in the dose unit.

[0169] As used herein, "a pharmacodynamic (PD) profile" refers to a
profile of the efficacy of a drug in a patient (or subject or user),
which is characterized by PD parameters. "PD parameters" include "drug
Emax" (the maximum drug efficacy), "drug EC50" (the concentration of drug
at 50% of the Emax), and side effects.

[0170]FIG. 1 is a schematic illustrating an example of the effect of
increasing inhibitor concentrations upon the PK parameter drug Cmaxfor a
fixed dose of prodrug. At low concentrations of inhibitor, there may be
no detectable effect on drug release, as illustrated by the plateau
portion of the plot of drug Cmax(Y axis) versus inhibitor concentration
(X axis). As inhibitor concentration increases, a concentration is
reached at which drug release from prodrug is attenuated, causing a
decrease in, or suppression of, drug Cmax. Thus, the effect of inhibitor
upon a prodrug PK parameter for a dose unit of the present disclosure can
range from undetectable, to moderate, to complete inhibition (i.e., no
detectable drug release).

[0171] A dose unit can be adapted to provide for a desired PK profile
(e.g., a concentration-time PK profile) following ingestion of a single
dose. A dose unit can be adapted to provide for a desired PK profile
(e.g., a concentration-dose PK profile) following ingestion of multiple
dose units (e.g., at least 2, at least 3, at least 4 or more dose units).

Dose Units Providing Modified PK Profiles

[0172] A combination of a prodrug and an inhibitor in a dose unit can
provide a desired (or "pre-selected") PK profile (e.g., a
concentration-time PK profile) following ingestion of a single dose. The
PK profile of such a dose unit can be characterized by one or more of a
pre-selected drug Cmax, a pre-selected drug Tmax or a pre-selected drug
exposure. The PK profile of the dose unit can be modified compared to a
PK profile achieved from the equivalent dosage of prodrug in the absence
of inhibitor (i.e., a dose that is the same as the dose unit except that
it lacks inhibitor).

[0173] A modified PK profile can have a decreased PK parameter value
relative to a reference PK parameter value (e.g., a PK parameter value of
a PK profile following ingestion of a dosage of prodrug that is
equivalent to a dose unit except without inhibitor). For example, a dose
unit can provide for a decreased drug Cmax, decreased drug exposure,
and/or delayed drug Tmax.

[0174]FIG. 2 presents schematic graphs showing examples of modified
concentration-time PK profiles of a single dose unit. Panel A is a
schematic of drug concentration in blood or plasma (Y axis) following a
period of time (X axis) after ingestion of prodrug in the absence or
presence of inhibitor. The solid, top line in Panel A provides an example
of drug concentration following ingestion of prodrug without inhibitor.
The dashed, lower line in Panel A represents drug concentration following
ingestion of the same dose of prodrug with inhibitor. Ingestion of
inhibitor with prodrug provides for a decreased drug Cmaxrelative to the
drug Cmaxthat results from ingestion of the same amount of prodrug in the
absence of inhibitor. Panel A also illustrates that the total drug
exposure following ingestion of prodrug with inhibitor is also decreased
relative to ingestion of the same amount of prodrug without inhibitor.

[0175] Panel B of FIG. 2 provides another example of a dose unit having a
modified concentration-time PK profile. As in Panel A, the solid top line
represents drug concentration over time in blood or plasma following
ingestion of prodrug without inhibitor, while the dashed lower line
represents drug concentration following ingestion of the same amount of
prodrug with inhibitor. In this example, the dose unit provides a PK
profile having a decreased drug Cmax, decreased drug exposure, and a
delayed drug Tmax (i.e., decreased (1/drug Tmax) relative to ingestion of
the same dose of prodrug without inhibitor.

[0176] Panel C of FIG. 2 provides another example of a dose unit having a
modified concentration-time PK profile. As in Panel A, the solid line
represents drug concentration over time in blood or plasma following
ingestion of prodrug without inhibitor, while the dashed line represents
drug concentration following ingestion of the same amount of prodrug with
inhibitor. In this example, the dose unit provides a PK profile having a
delayed drug Tmax (i.e., decreased (1/drug Tmax) relative to ingestion of
the same dose of prodrug without inhibitor.

[0177] Dose units that provide for a modified PK profile (e.g., a
decreased drug Cmaxand/or delayed drug Tmax as compared to, a PK profile
of drug or a PK profile of prodrug without inhibitor), find use in
tailoring of drug dose according to a patient's needs (e.g., through
selection of a particular dose unit and/or selection of a dosage
regimen), reduction of side effects, and/or improvement in patient
compliance (as compared to side effects or patient compliance associated
with drug or with prodrug without inhibitor). As used herein, "patient
compliance" refers to whether a patient follows the direction of a
clinician (e.g., a physician) including ingestion of a dose that is
neither significantly above nor significantly below that prescribed. Such
dose units also reduce the risk of misuse, abuse or overdose by a patient
as compared to such risk(s) associated with drug or prodrug without
inhibitor. For example, dose units with a decreased drug Cmaxprovide less
reward for ingestion than does a dose of the same amount of drug, and/or
the same amount of prodrug without inhibitor.

[0178] A dose unit of the present disclosure can be adapted to provide for
a desired PK profile (e.g., a concentration-time PK profile or
concentration-dose PK profile) following ingestion of multiples of a dose
unit (e.g., at least 2, at least 3, at least 4, or more dose units). A
concentration-dose PK profile refers to the relationship between a
selected PK parameter and a number of single dose units ingested. Such a
profile can be dose proportional, linear (a linear PK profile) or
nonlinear (a nonlinear PK profile). A modified concentration-dose PK
profile can be provided by adjusting the relative amounts of prodrug and
inhibitor contained in a single dose unit and/or by using a different
prodrug and/or inhibitor.

[0179]FIG. 3 provides schematics of examples of concentration-dose PK
profiles (exemplified by drug Cmax, Y axis) that can be provided by
ingestion of multiples of a dose unit (X axis) of the present disclosure.
Each profile can be compared to a concentration-dose PK profile provided
by increasing doses of drug alone, where the amount of drug in the blood
or plasma from one dose represents a therapeutically effective amount
equivalent to the amount of drug released into the blood or plasma by one
dose unit of the disclosure. Such a "drug alone" PK profile is typically
dose proportional, having a forty-five degree angle positive linear
slope. It is also to be appreciated that a concentration-dose PK profile
resulting from ingestion of multiples of a dose unit of the disclosure
can also be compared to other references, such as a concentration-dose PK
profile provided by ingestion of an increasing number of doses of prodrug
without inhibitor wherein the amount of drug released into the blood or
plasma by a single dose of prodrug in the absence of inhibitor represents
a therapeutically effective amount equivalent to the amount of drug
released into the blood or plasma by one dose unit of the disclosure.

[0180] As illustrated by the relationship between prodrug and inhibitor
concentration in FIG. 1, a dose unit can include inhibitor in an amount
that does not detectably affect drug release following ingestion.
Ingestion of multiples of such a dose unit can provide a
concentration-dose PK profile such that the relationship between number
of dose units ingested and PK parameter value is linear with a positive
slope, which is similar to, for example, a dose proportional PK profile
of increasing amounts of prodrug alone. Panel A of FIG. 3 depicts such a
profile. Dose units that provide a concentration-dose PK profile having
such an undetectable change in drug Cmaxin vivo compared to the profile
of prodrug alone can find use in thwarting enzyme conversion of prodrug
from a dose unit that has sufficient inhibitor to reduce or prevent in
vitro cleavage of the enzyme-cleavable prodrug by its respective enzyme.

[0181] Panel B in FIG. 3 represents a concentration-dose PK profile such
that the relationship between the number of dose units ingested and a PK
parameter value is linear with positive slope, where the profile exhibits
a reduced slope relative to panel A. Such a dose unit provides a profile
having a decreased PK parameter value (e.g., drug Cmax) relative to a
reference PK parameter value exhibiting dose proportionality.

[0182] Concentration-dose PK profiles following ingestion of multiples of
a dose unit can be non-linear. Panel C in FIG. 3 represents an example of
a non-linear, biphasic concentration-dose PK profile. In this example,
the biphasic concentration-dose PK profile contains a first phase over
which the concentration-dose PK profile has a positive rise, and then a
second phase over which the relationship between number of dose units
ingested and a PK parameter value (e.g., drug Cmax) is relatively flat
(substantially linear with zero slope). For such a dose unit, for
example, drug Cmaxcan be increased for a selected number of dose units
(e.g., 2, 3, or 4 dose units). However, ingestion of additional dose
units does not provide for a significant increase in drug Cmax.

[0183] Panel D in FIG. 3 represents another example of a non-linear,
biphasic concentration-dose PK profile. In this example, the biphasic
concentration-dose PK profile is characterized by a first phase over
which the concentration-dose PK profile has a positive rise and a second
phase over which the relationship between number of dose units ingested
and a PK parameter value (e.g., drug Cmax) declines. Dose units that
provide this concentration-dose PK profile provide for an increase in
drug Cmaxfor a selected number of ingested dose units (e.g., 2, 3, or 4
dose units). However, ingestion of further additional dose units does not
provide for a significant increase in drug Cmaxand instead provides for
decreased drug Cmax.

[0184] Panel E in FIG. 3 represents a concentration-dose PK profile in
which the relationship between the number of dose units ingested and a PK
parameter (e.g., drug Cmax) is linear with zero slope. Such dose units do
not provide for a significant increase or decrease in drug Cmax with
ingestion of multiples of dose units.

[0185] Panel F in FIG. 3 represents a concentration-dose PK profile in
which the relationship between number of dose units ingested and a PK
parameter value (e.g., drug Cmax) is linear with a negative slope. Thus
drug Cmaxdecreases as the number of dose units ingested increases.

[0186] Dose units that provide for concentration-dose PK profiles when
multiples of a dose unit are ingested find use in tailoring of a dosage
regimen to provide a therapeutic level of released drug while reducing
the risk of overdose, misuse, or abuse. Such reduction in risk can be
compared to a reference, e.g., to administration of drug alone or prodrug
alone. In one embodiment, risk is reduced compared to administration of a
drug or prodrug that provides a proportional concentration-dose PK
profile. A dose unit that provides for a concentration-dose PK profile
can reduce the risk of patient overdose through inadvertent ingestion of
dose units above a prescribed dosage. Such a dose unit can reduce the
risk of patient misuse (e.g., through self-medication). Such a dose unit
can discourage abuse through deliberate ingestion of multiple dose units.
For example, a dose unit that provides for a biphasic concentration-dose
PK profile can allow for an increase in drug release for a limited number
of dose units ingested, after which an increase in drug release with
ingestion of more dose units is not realized. In another example, a dose
unit that provides for a concentration-dose PK profile of zero slope can
allow for retention of a similar drug release profile regardless of the
number of dose units ingested.

[0187] Ingestion of multiples of a dose unit can provide for adjustment of
a PK parameter value relative to that of ingestion of multiples of the
same dose (either as drug alone or as a prodrug) in the absence of
inhibitor such that, for example, ingestion of a selected number (e.g.,
2, 3, 4 or more) of a single dose unit provides for a decrease in a PK
parameter value compared to ingestion of the same number of doses in the
absence of inhibitor.

[0188] Pharmaceutical compositions include those having an inhibitor to
provide for protection of a therapeutic compound from degradation in the
GI tract. Inhibitor can be combined with a drug (i.e., not a prodrug) to
provide for protection of the drug from degradation in the GI system.

[0189] In this example, the composition of inhibitor and drug provide for
a modified PK profile by increasing a PK parameter. Inhibitor can also be
combined with a prodrug that is susceptible to degradation by a GI enzyme
and has a site of action outside the GI tract. In this composition, the
inhibitor protects ingested prodrug in the GI tract prior to its
distribution outside the GI tract and cleavage at a desired site of
action.

Methods Used to Define Relative Amounts of Prodrug and Inhibitor in a Dose
Unit

[0190] Dose units that provide for a desired PK profile, such as a desired
concentration-time PK profile and/or a desired concentration-dose PK
profile, can be made by combining a prodrug and an inhibitor in a dose
unit in relative amounts effective to provide for release of drug that
provides for a desired drug PK profile following ingestion by a patient.

[0191] Prodrugs can be selected as suitable for use in a dose unit by
determining the trypsin-mediated drug release competency of the prodrug.
This can be accomplished in vitro, in vivo or ex vivo.

[0192] In vitro assays can be conducted by combining a prodrug with
trypsin in a reaction mixture. Trypsin can be provided in the reaction
mixture in an amount sufficient to catalyze cleavage of the prodrug.
Assays are conducted under suitable conditions, and optionally may be
under conditions that mimic those found in a GI tract of a subject, e.g.,
human. "Prodrug conversion" refers to release of drug from prodrug.
Prodrug conversion can be assessed by detecting a level of a product of
prodrug conversion (e.g., released drug) and/or by detecting a level of
prodrug that is maintained in the presence of trypsin. Prodrug conversion
can also be assessed by detecting the rate at which a product of prodrug
conversion occurs or the rate at which prodrug disappears. An increase in
released drug, or a decrease in prodrug, indicate prodrug conversion has
occurred. Prodrugs that exhibit an acceptable level of prodrug conversion
in the presence of trypsin within an acceptable period of time are
suitable for use in a dose unit in combination with a trypsin inhibitor.

[0193] In vivo assays can assess the suitability of a prodrug for use in a
dose unit by administration of the prodrug to an animal (e.g., a human or
non-human animal, e.g., rat, dog, pig, etc.). Such administration can be
enteral (e.g., oral administration). Prodrug conversion can be detected
by, for example, detecting a product of prodrug conversion (e.g.,
released drug or a metabolite of released drug) or detecting prodrug in
blood or plasma of the animal at a desired time point(s) following
administration.

[0194] Ex vivo assays, such as a gut loop or inverted gut loop assay, can
assess the suitability of a prodrug for use in a dose unit by, for
example, administration of the prodrug to a ligated section of the
intestine of an animal. Prodrug conversion can be detected by, for
example, detecting a product of prodrug conversion (e.g., released drug
or a metabolite of released drug) or detecting prodrug in the ligated gut
loop of the animal at a desired time point(s) following administration.

[0195] Inhibitors are generally selected based on, for example, activity
in interacting with trypsin that mediates release of drug from a prodrug
with which the inhibitor is to be co-dosed. Such assays can be conducted
in the presence of enzyme either with or without prodrug. Inhibitors can
also be selected according to properties such as half-life in the GI
system, potency, avidity, affinity, molecular size and/or enzyme
inhibition profile (e.g., steepness of inhibition curve in an enzyme
activity assay, inhibition initiation rate). Inhibitors for use in
prodrug-inhibitor combinations can be selected through use of in vitro,
in vivo and/or ex vivo assays.

[0196] One embodiment is a method for identifying a prodrug and a trypsin
inhibitor suitable for formulation in a dose unit wherein the method
comprises combining a prodrug (e.g., Compound PC-5), a trypsin inhibitor,
and trypsin in a reaction mixture and detecting prodrug conversion. Such
a combination is tested for an interaction between the prodrug, inhibitor
and enzyme, i.e., tested to determine how the inhibitor will interact
with the enzyme that mediates enzymatically-controlled release of the
drug from the prodrug. In one embodiment, a decrease in prodrug
conversion in the presence of the trypsin inhibitor as compared to
prodrug conversion in the absence of the trypsin inhibitor indicates the
prodrug and trypsin inhibitor are suitable for formulation in a dose
unit. Such a method can be an in vitro assay.

[0197] One embodiment is a method for identifying a prodrug and a trypsin
inhibitor suitable for formulation in a dose unit wherein the method
comprises administering to an animal a prodrug (e.g., Compound PC-5) and
a trypsin inhibitor and detecting prodrug conversion. In one embodiment,
a decrease in prodrug conversion in the presence of the trypsin inhibitor
as compared to prodrug conversion in the absence of the trypsin inhibitor
indicates the prodrug and trypsin inhibitor are suitable for formulation
in a dose unit. Such a method can be an in vivo assay; for example, the
prodrug and trypsin inhibitor can be administered orally. Such a method
can also be an ex vivo assay; for example, the prodrug and trypsin
inhibitor can be administered orally or to a tissue, such as an
intestine, that is at least temporarily exposed. Detection can occur in
the blood or plasma or respective tissue. As used herein, tissue refers
to the tissue itself and can also refer to contents within the tissue.

[0198] One embodiment is a method for identifying a prodrug and a trypsin
inhibitor suitable for formulation in a dose unit wherein the method
comprises administering a prodrug and a trypsin inhibitor to an animal
tissue that has removed from an animal and detecting prodrug conversion.
In one embodiment, a decrease in prodrug conversion in the presence of
the trypsin inhibitor as compared to prodrug conversion in the absence of
the trypsin inhibitor indicates the prodrug and trypsin inhibitor are
suitable for formulation in a dose unit.

[0199] In vitro assays can be conducted by combining a prodrug, a trypsin
inhibitor and trypsin in a reaction mixture. Trypsin can be provided in
the reaction mixture in an amount sufficient to catalyze cleavage of the
prodrug, and assays conducted under suitable conditions, optionally under
conditions that mimic those found in a GI tract of a subject, e.g.,
human. Prodrug conversion can be assessed by detecting a level of a
product of prodrug conversion (e.g., released drug) and/or by detecting a
level of prodrug maintained in the presence of trypsin. Prodrug
conversion can also be assessed by detecting the rate at which a product
of prodrug conversion occurs or the rate at which prodrug disappears.
Prodrug conversion that is modified in the presence of inhibitor as
compared to a level of prodrug conversion in the absence of inhibitor
indicates the inhibitor is suitable for attenuation of prodrug conversion
and for use in a dose unit. Reaction mixtures having a fixed amount of
prodrug and increasing amounts of inhibitor, or a fixed amount of
inhibitor and increasing amounts of prodrug, can be used to identify
relative amounts of prodrug and inhibitor which provide for a desired
modification of prodrug conversion.

[0200] In vivo assays can assess combinations of prodrugs and inhibitors
by co-dosing of prodrug and inhibitor to an animal. Such co-dosing can be
enteral. "Co-dosing" refers to administration of prodrug and inhibitor as
separate doses or a combined dose (i.e., in the same formulation).
Prodrug conversion can be detected by, for example, detecting a product
of prodrug conversion (e.g., released drug or drug metabolite) or
detecting prodrug in blood or plasma of the animal at a desired time
point(s) following administration. Combinations of prodrug and inhibitor
can be identified that provide for a prodrug conversion level that yields
a desired PK profile as compared to, for example, prodrug without
inhibitor.

[0201] Combinations of relative amounts of prodrug and inhibitor that
provide for a desired PK profile can be identified by dosing animals with
a fixed amount of prodrug and increasing amounts of inhibitor, or with a
fixed amount of inhibitor and increasing amounts of prodrug. One or more
PK parameters can then be assessed, e.g., drug Cmax, drug Tmax, and drug
exposure. Relative amounts of prodrug and inhibitor that provide for a
desired PK profile are identified as amounts of prodrug and inhibitor for
use in a dose unit. The PK profile of the prodrug and inhibitor
combination can be, for example, characterized by a decreased PK
parameter value relative to prodrug without inhibitor. A decrease in the
PK parameter value of an inhibitor-to-prodrug combination (e.g., a
decrease in drug Cmax, a decrease in 1/drug Tmax (i.e., a delay in drug
Tmax) or a decrease in drug exposure) relative to a corresponding PK
parameter value following administration of prodrug without inhibitor can
be indicative of an inhibitor-to-prodrug combination that can provide a
desired PK profile. Assays can be conducted with different relative
amounts of inhibitor and prodrug.

[0202] In vivo assays can be used to identify combinations of prodrug and
inhibitor that provide for dose units that provide for a desired
concentration-dose PK profile following ingestion of multiples of the
dose unit (e.g., at least 2, at least 3, at least 4 or more). Ex vivo
assays can be conducted by direct administration of prodrug and inhibitor
into a tissue and/or its contents of an animal, such as the intestine,
including by introduction by injection into the lumen of a ligated
intestine (e.g., a gut loop, or intestinal loop, assay, or an inverted
gut assay). An ex vivo assay can also be conducted by excising a tissue
and/or its contents from an animal and introducing prodrug and inhibitor
into such tissues and/or contents.

[0203] For example, a dose of prodrug that is desired for a single dose
unit is selected (e.g., an amount that provides an efficacious plasma
drug level). A multiple of single dose units for which a relationship
between that multiple and a PK parameter to be tested is then selected.
For example, if a concentration-dose PK profile is to be designed for
ingestion of 2, 3, 4, 5, 6, 7, 8, 9 or 10 dose units, then the amount of
prodrug equivalent to ingestion of that same number of dose units is
determined (referred to as the "high dose"). The multiple of dose units
can be selected based on the number of ingested pills at which drug
Cmaxis modified relative to ingestion of the single dose unit. If, for
example, the profile is to provide for abuse deterrence, then a multiple
of 10 can be selected, for example. A variety of different inhibitors
(e.g., from a panel of inhibitors) can be tested using different relative
amounts of inhibitor and prodrug. Assays can be used to identify suitable
combination(s) of inhibitor and prodrug to obtain a single dose unit that
is therapeutically effective, wherein such a combination, when ingested
as a multiple of dose units, provides a modified PK parameter compared to
ingestion of the same multiple of drug or prodrug alone (wherein a single
dose of either drug or prodrug alone releases into blood or plasma the
same amount of drug as is released by a single dose unit).

[0204] Increasing amounts of inhibitor are then co-dosed to animals with
the high dose of prodrug. The dose level of inhibitor that provides a
desired drug Cmaxfollowing ingestion of the high dose of prodrug is
identified and the resultant inhibitor-to-prodrug ratio determined.

[0205] Prodrug and inhibitor are then co-dosed in amounts equivalent to
the inhibitor-to-prodrug ratio that provided the desired result at the
high dose of prodrug. The PK parameter value of interest (e.g., drug
Cmax) is then assessed. If a desired PK parameter value results following
ingestion of the single dose unit equivalent, then single dose units that
provide for a desired concentration-dose PK profile are identified. For
example, where a zero dose linear profile is desired, the drug
Cmaxfollowing ingestion of a single dose unit does not increase
significantly following ingestion of a multiple number of the single dose
units.

Methods for Manufacturing, Formulating, and Packaging Dose Units

[0206] Dose units of the present disclosure can be made using
manufacturing methods available in the art and can be of a variety of
forms suitable for enteral (including oral, buccal and sublingual)
administration, for example as a tablet, capsule, thin film, powder,
suspension, solution, syrup, dispersion or emulsion. The dose unit can
contain components conventional in pharmaceutical preparations, e.g. one
or more carriers, binders, lubricants, excipients (e.g., to impart
controlled release characteristics), pH modifiers, flavoring agents
(e.g., sweeteners), bulking agents, coloring agents or further active
agents. Dose units of the present disclosure can include can include an
enteric coating or other component(s) to facilitate protection from
stomach acid, where desired.

[0207] Dose units can be of any suitable size or shape. The dose unit can
be of any shape suitable for enteral administration, e.g., ellipsoid,
lenticular, circular, rectangular, cylindrical, and the like.

[0208] Dose units provided as dry dose units can have a total weight of
from about 1 microgram to about 1 gram, and can be from about 5
micrograms to 1.5 grams, from about 50 micrograms to 1 gram, from about
100 micrograms to 1 gram, from 50 micrograms to 750 milligrams, and may
be from about 1 microgram to 2 grams.

[0209] Dose units can comprise components in any relative amounts. For
example, dose units can be from about 0.1% to 99% by weight of active
ingredients (i.e., prodrug and inhibitor) per total weight of dose unit
(0.1% to 99% total combined weight of prodrug and inhibitor per total
weight of single dose unit). In some embodiments, dose units can be from
10% to 50%, from 20% to 40%, or about 30% by weight of active ingredients
per total weight dose unit.

[0210] Dose units can be provided in a variety of different forms and
optionally provided in a manner suitable for storage. For example, dose
units can be disposed within a container suitable for containing a
pharmaceutical composition. The container can be, for example, a bottle
(e.g., with a closure device, such as a cap), a blister pack (e.g., which
can provide for enclosure of one or more dose units per blister), a vial,
flexible packaging (e.g., sealed Mylar or plastic bags), an ampule (for
single dose units in solution), a dropper, thin film, a tube and the
like.

[0211] Containers can include a cap (e.g., screw cap) that is removably
connected to the container over an opening through which the dose units
disposed within the container can be accessed.

[0212] Containers can include a seal which can serve as a tamper-evident
and/or tamper-resistant element, which seal is disrupted upon access to a
dose unit disposed within the container. Such seal elements can be, for
example, a frangible element that is broken or otherwise modified upon
access to a dose unit disposed within the container. Examples of such
frangible seal elements include a seal positioned over a container
opening such that access to a dose unit within the container requires
disruption of the seal (e.g., by peeling and/or piercing the seal).
Examples of frangible seal elements include a frangible ring disposed
around a container opening and in connection with a cap such that the
ring is broken upon opening of the cap to access the dose units in the
container.

[0213] Dry and liquid dose units can be placed in a container (e.g.,
bottle or package, e.g., a flexible bag) of a size and configuration
adapted to maintain stability of dose units over a period during which
the dose units are dispensed into a prescription. For example, containers
can be sized and configured to contain 10, 20, 30, 40, 50, 60, 70, 80,
90, 100 or more single dry or liquid dose units. The containers can be
sealed or resealable. The containers can packaged in a carton (e.g., for
shipment from a manufacturer to a pharmacy or other dispensary). Such
cartons can be boxes, tubes, or of other configuration, and may be made
of any material (e.g., cardboard, plastic, and the like). The packaging
system and/or containers disposed therein can have one or more affixed
labels (e.g., to provide information such as lot number, dose unit type,
manufacturer, and the like).

[0214] The container can include a moisture barrier and/or light barrier,
e.g., to facilitate maintenance of stability of the active ingredients in
the dose units contained therein. Where the dose unit is a dry dose unit,
the container can include a desiccant pack which is disposed within the
container. The container can be adapted to contain a single dose unit or
multiples of a dose unit. The container can include a dispensing control
mechanism, such as a lock out mechanism that facilitates maintenance of
dosing regimen.

[0215] The dose units can be provided in solid or semi-solid form, and can
be a dry dose unit. "Dry dose unit" refers to a dose unit that is in
other than in a completely liquid form. Examples of dry dose units
include, for example, tablets, capsules (e.g., solid capsules, capsules
containing liquid), thin film, microparticles, granules, powder and the
like. Dose units can be provided as liquid dose units, where the dose
units can be provided as single or multiple doses of a formulation
containing prodrug and inhibitor in liquid form. Single doses of a dry or
liquid dose unit can be disposed within a sealed container, and sealed
containers optionally provided in a packaging system, e.g., to provide
for a prescribed number of doses, to provide for shipment of dose units,
and the like.

[0216] Dose units can be formulated such that the prodrug and inhibitor
are present in the same carrier, e.g., solubilized or suspended within
the same matrix. Alternatively, dose units can be composed of two or more
portions, where the prodrug and inhibitor can be provided in the same or
different portions, and can be provided in adjacent or non-adjacent
portions.

[0217] Dose units can be provided in a container in which they are
disposed, and may be provided as part of a packaging system (optionally
with instructions for use). For example, dose units containing different
amounts of prodrug can be provided in separate containers, which
containers can be disposed with in a larger container (e.g., to
facilitate protection of dose units for shipment). For example, one or
more dose units as described herein can be provided in separate
containers, where dose units of different composition are provided in
separate containers, and the separate containers disposed within package
for dispensing.

[0218] In another example, dose units can be provided in a
double-chambered dispenser where a first chamber contains a prodrug
formulation and a second chamber contains an inhibitor formulation. The
dispenser can be adapted to provide for mixing of a prodrug formulation
and an inhibitor formulation prior to ingestion. For example, the two
chambers of the dispenser can be separated by a removable wall (e.g.,
frangible wall) that is broken or removed prior to administration to
allow mixing of the formulations of the two chambers. The first and
second chambers can terminate into a dispensing outlet, optionally
through a common chamber. The formulations can be provided in dry or
liquid form, or a combination thereof. For example, the formulation in
the first chamber can be liquid and the formulation in the second chamber
can be dry, both can be dry, or both can be liquid.

[0219] Dose units that provide for controlled release of prodrug, of
inhibitor, or of both prodrug and inhibitor are contemplated by the
present disclosure, where "controlled release" refers to release of one
or both of prodrug and inhibitor from the dose unit over a selected
period of time and/or in a pre-selected manner.

Methods of Use of Dose Units

[0220] Dose units are advantageous because they find use in methods to
reduce side effects and/or improve tolerability of drugs to patients in
need thereof by, for example, limiting a PK parameter as disclosed
herein. The present disclosure thus provides methods to reduce side
effects by administering a dose unit of the present disclosure to a
patient in need so as to provide for a reduction of side effects as
compared to those associated with administration of drug and/or as
compared to administration of prodrug without inhibitor. The present
disclosure also provides methods to improve tolerability of drugs by
administering a dose unit of the present disclosure to a patient in need
so as to provide for improvement in tolerability as compared to
administration of drug and/or as compared to administration of prodrug
without inhibitor.

[0221] Dose units find use in methods for increasing patient compliance of
a patient with a therapy prescribed by a clinician, where such methods
involve directing administration of a dose unit described herein to a
patient in need of therapy so as to provide for increased patient
compliance as compared to a therapy involving administration of drug
and/or as compared to administrations of prodrug without inhibitor. Such
methods can help increase the likelihood that a clinician-specified
therapy occurs as prescribed.

[0222] Dose units can provide for enhanced patient compliance and
clinician control. For example, by limiting a PK parameter (e.g., such as
drug Cmaxor drug exposure) when multiples (e.g., two or more, three or
more, or four or more) dose units are ingested, a patient requiring a
higher dose of drug must seek the assistance of a clinician. The dose
units can provide for control of the degree to which a patient can
readily "self-medicate", and further can provide for the patient to
adjust dose to a dose within a permissible range. Dose units can provide
for reduced side effects, by for example, providing for delivery of drug
at an efficacious dose but with a modified PK profile over a period of
treatment, e.g., as defined by a decreased PK parameter (e.g., decreased
drug Cmax, decreased drug exposure).

[0223] Dose units find use in methods to reduce the risk of unintended
overdose of drug that can follow ingestion of multiple doses taken at the
same time or over a short period of time. Such methods of the present
disclosure can provide for reduction of risk of unintended overdose as
compared to risk of unintended overdose of drug and/or as compared to
risk of unintended overdose of prodrug without inhibitor. Such methods
involve directing administration of a dosage described herein to a
patient in need of drug released by conversion of the prodrug. Such
methods can help avoid unintended overdosing due to intentional or
unintentional misuse of the dose unit.

[0224] The present disclosure provides methods to reduce misuse and abuse
of a drug, as well as to reduce risk of overdose, that can accompany
ingestion of multiples of doses of a drug, e.g., ingested at the same
time. Such methods generally involve combining in a dose unit a prodrug
and a trypsin inhibitor that mediates release of drug from the prodrug,
where the inhibitor is present in the dose unit in an amount effective to
attenuate release of drug from the prodrug, e.g., following ingestion of
multiples of dose units by a patient. Such methods provide for a modified
concentration-dose PK profile while providing therapeutically effective
levels from a single dose unit, as directed by the prescribing clinician.
Such methods can provide for, for example, reduction of risks that can
accompany misuse and/or abuse of a prodrug, particularly where conversion
of the prodrug provides for release of a narcotic or other drug of abuse
(e.g., opioid). For example, when the prodrug provides for release of a
drug of abuse, dose units can provide for reduction of reward that can
follow ingestion of multiples of dose units of a drug of abuse.

[0225] Dose units can provide clinicians with enhanced flexibility in
prescribing drug. For example, a clinician can prescribe a dosage regimen
involving different dose strengths, which can involve two or more
different dose units of prodrug and inhibitor having different relative
amounts of prodrug, different amounts of inhibitor, or different amounts
of both prodrug and inhibitor. Such different strength dose units can
provide for delivery of drug according to different PK parameters (e.g.,
drug exposure, drug Cmax, and the like as described herein). For example,
a first dose unit can provide for delivery of a first dose of drug
following ingestion, and a second dose unit can provide for delivery of a
second dose of drug following ingestion. The first and second prodrug
doses of the dose units can be different strengths, e.g., the second dose
can be greater than the first dose. A clinician can thus prescribe a
collection of two or more, or three or more dose units of different
strengths, which can be accompanied by instructions to facilitate a
degree of self-medication, e.g., to increase delivery of an opioid drug
according to a patient's needs to treat pain.

Thwarting Tampering by Trypsin Mediated Release of Hydromorphone from
Prodrug

[0226] The disclosure provides for a composition comprising Compound PC-5
and a trypsin inhibitor that reduces drug abuse potential. A trypsin
inhibitor can thwart the ability of a user to apply trypsin to effect the
release of hydromorphone from the phenol-modified hydromorphone prodrug,
Compound PC-5, in vitro. For example, if an abuser attempts to incubate
trypsin with a composition of the embodiments that includes Compound PC-5
and a trypsin inhibitor, the trypsin inhibitor can reduce the action of
the added trypsin, thereby thwarting attempts to release hydromorphone
for purposes of abuse.

EXAMPLES

[0227] The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and description of
how to make and use the embodiments, and are not intended to limit the
scope of what the inventors regard as their invention nor are they
intended to represent that the experiments below are all or the only
experiments performed. Efforts have been made to ensure accuracy with
respect to numbers used (e.g. amounts, temperature, etc.) but some
experimental errors and deviations should be accounted for. Unless
indicated otherwise, parts are parts by weight, molecular weight is
weight average molecular weight, temperature is in degrees Celsius, and
pressure is at or near atmospheric. Standard abbreviations may be used.

[0229] To a solution of Fmoc-Arg(Pbf)-OH 1 (25.0 g, 38.5 mmol) in DMF (200
mL) at room temperature was added DIEA (13.41 mL, 77.1 mmol). After
stiffing at room temperature for 10 min, the reaction mixture was cooled
to ˜5° C. To the reaction mixture was added HATU (16.11 g,
42.4 mmol) in portions and stirred for 20 min and a solution of
tert-butyl-1-piperazine carboxylate (7.18 g, 38.5 mmol) in DMF (50 mL)
was added dropwise. The reaction mixture was stirred at ˜5°
C. for 5 min. The mixture reaction was then allowed to warm to room
temperature and stirred for 2 h. Solvent was removed in vacuo and the
residue was dissolved in EtOAc (500 mL), washed with water (2×750
mL), 1% H2SO4 (300 mL) and brine (750 mL). The organic layer
was separated, dried over Na2SO4 and solvent removed in vacuo
to a total volume of 100 mL. Compound A was taken to the next step as
EtOAc solution (100 mL). LC-MS [M+H] 817.5
(C43H56N6O8S+H, calc: 817.4).

[0230] To a solution of compound A (46.2 mmol) in EtOAc (175 mL) at room
temperature was added piperidine (4.57 mL, 46.2 mmol) and the reaction
mixture was stirred for 18 h at room temperature. Next the solvent was
removed in vacuo and the resulting residue dissolved in minimum amount of
EtOAc (˜50 mL) and hexane (˜1 L) was added. The precipitated
crude product was filtered off and recrystallised again with EtOAc
(˜30 mL) and hexane (˜750 mL). The precipitate was filtered
off, washed with hexane and dried in vacuo to afford compound B (28.0 g,
46.2 mmol). LC-MS [M+H] 595.4 (C28H46N6O6S+H, calc:
595.3). Compound B was used without further purification.

[0237] A solution of compound F (20.2 g, 30.2 mmol) in dichloromethane (90
mL) was treated with 4.0 N HCl in 1,4-dioxane (90 mL, 363.3 mmol) and
stirred at room temperature for 2 h. Next most of the dichloromethane
(˜90%) was removed in vacuo and Et2O (˜1 L) was added.
The resultant precipitate was filtered off and washed with Et2O and
dried in vacuo to afford compound G (17.8 g, 30.2 mmol). LC-MS [M+H]
567.8 (C26H42N6O6S+H, calc: 567.8). Compound G was
used without further purification.

[0242] To a solution of compound I (4.7 g, 7.07 mmol) in dichloromethane
(25 mL) was added 4N HCl in dioxane (25.0 mL, 84.84 mmol), and the
reaction mixture was stirred at room temperature for 2 h. The reaction
mixture was concentrated in vacuo to ˜20 mL of solvent, and then
diluted with diethyl ether (250 mL) to produce a white fine precipitate.
The reaction mixture was stirred for 1 h and the solid was washed with
ether (50 mL) and dried in vacuo overnight to give compound J (4.3 g,
7.07 mmol) as a fine powder. LC-MS [M+H] 566.5
(C27H43N5O6S+H, calc: 566.7). Compound J was used
without further purification.

[0243] To a solution of compound J (1.1 g, 1.6 mmol) and NaOH (260 mg, 5.9
mmol) in a mixture of THF (5 mL) and water (3 mL) was added a solution of
2-naphthalosulfonyl chloride (0.91 g, 2.5 mmol) in THF (10 mL) dropwise
with stirring at ˜5° C. The reaction mixture was stirred at
room temperature for 1 h, then diluted with water (5 mL). Aqueous 1N HCl
(5 mL) was added to obtain pH ˜3. Additional water was added (20
mL), and the product was extracted with ethyl acetate (3×50 mL).
The organic layer was removed and then washed with 2% aqueous sodium
bicarbonate (50 mL), water (50 mL) and brine (50 mL). The extract was
dried over anhydrous sodium sulfate, filtered, and was evaporated in
vacuo. The formed oily product was dried in vacuo overnight to give
compound K (1.3 g, 1.6 mmol) as an oily foaming solid. LC-MS [M+H] 756.5
(C37H49N5O8S2+H, calc: 756.7). Compound K was
used without further purification.

[0246] To a solution of compound J (1.0 g, 1.6 mmol) and NaOH (420.0 mg,
10.4 mmol) in a mixture of THF (5 mL) and water (4 mL) was added a
solution of 2,4,6-triisopropyl-benzenesulfonyl chloride (2.4 g, 8.0 mmol)
dropwise with stirring and maintained at ˜5° C. The reaction
mixture was then stirred at room temperature for 1 h, monitoring the
reaction progress, then diluted with water (20 mL), and acidified with
aqueous 1 N HCl (5 mL) to pH ˜3. Additional water was added (30
mL), and the product was extracted with EtOAc (3×50 mL). The
organic layer was washed with 2% aqueous sodium bicarbonate (50 mL),
water (50 mL) and brine (50 mL). The organic layer was dried over
anhydrous sodium sulfate, filtered, and was evaporated in vacuo. Formed
oily residue was dried in a vacuo overnight to give compound N (1.0 g,
1.2 mmol) as an oily material. LC-MS [M+H] 832.8
(C42H65N5O8S2+H, calc: 832.7). Compound N was
used without further purification.

[0252] Compound 107, i.e., 3-(4-carbamimidoylphenyl)-2-oxopropanoic acid
can be produced using methods known to those skilled in the art, such as
that described by Richter P et al, Pharmazie, 1977, 32, 216-220 and
references contained within. The purity of Compound 107 used herein was
estimated to be 76%, an estimate due low UV absorbance of this compound
via HPLC. Mass spec data: LC-MS [M+H] 207.0 (C10H10N2O3+H, calc: 207.1).

[0256] A solution of compound R (14.8 g, 20.0 mmol) and acetic anhydride
(5.7 mL, 60.0 mmol) in acetic acid (100 mL) was stirred at room
temperature for 45 min, and then solvent was evaporated in vacuo. The
resultant residue was dissolved in EtOAc (300 mL) and extracted with
water (2×75 mL) and brine (75 mL). The organic layer was then dried
over MgSO4, evaporated and dried in vacuo to provide compound S
(9.58 g, 18.2 mmol) as yellowish solid. LC-MS [M+H] 527.6
(C27H34N4O7+H, calc: 527.9). Compound S was used
without further purification.

[0258] A solution of D-homo-phenylalanine (10.0 g, 55.9 mmol) and NaOH
(3.35 g, 83.8 mmol) in a mixture of 1,4-dioxane (80 mL) and water (50 mL)
was cooled to ˜5° C., followed by alternate addition of
α-toluenesulfonyl chloride (16.0 g, 83.8 mmol; 5 portions by 3.2 g)
and 1.12 M NaOH (50 mL, 55.9 mmol; 5 portions by 10 mL) maintaining
pH>10. The reaction mixture was then acidified with 2% aq.
H2SO4 to a pH of about pH 2. The obtained solution was
extracted with EtOAc (2×200 mL). The obtained organic layer was
washed with water (3×75 mL), dried over MgSO4 and then the
solvent was evaporated in vacuo. The resultant residue was dried in vacuo
to provide compound U (12.6 g, 37.5 mmol) as white solid. LC-MS [M+H]
334.2 (C17H19NO4S+H, calc: 333.4). Compound U was used
without further purification.

[0263] A solution of compound QQ (24.6 g, 82.4 mmol) and DIEA (14.3 mL,
82.4 mmol) in THF (100 mL) was cooled to ˜5° C., followed by
the addition a solution of N-(benzyloxycarbonyl)succinimide (20.3 g, 81.6
mmol) in THF (75 mL) dropwise over 20 min. The temperature of the
reaction mixture was raised to room temperature and stirring was
continued for an additional 30 minutes (min). Solvents were evaporated
and the residue was dissolved in ethyl acetate (500 mL). The organic
layer was extracted with 5% aq. NaHCO3 (2×100 mL) and brine
(100 mL). The organic layer was evaporated to provide compound RR (24.9
g, 78.2 mmol) as a yellowish oil. LC-MS [M+H] 319.0
(C14H17F3N2O3+H, calc: 319.2). Compound RR was
used without further purification.

[0264] To a solution of compound RR (24.9 g, 78.2 mmol) in methanol (300
mL) was added a solution of LiOH (3.8 g, 156 mmol) in water (30 mL). The
reaction mixture was stirred at room temperature for 5 h. Next the
solvents were evaporated to 75% of initial volume followed by dilution
with water (200 mL). The solution was extracted with ethyl acetate (200
mL×2) and the organic layer was washed with brine (100 mL), dried
over MgSO4 and evaporated in vacuo. Residue was dissolved in ether
(200 mL) and treated with 2 N HCl/ether (200 mL). The formed precipitate
was filtered, washed with ether and dried in vacuo to provide the
hydrochloric salt of compound SS (12.1 g, 46.7 mmol) as a white solid.
LC-MS [M+H] 222.9 (C12H18N2O2+H, calc: 223.2). Purity
>95% (UV/254 nm).

[0266] Compound TT (18.5 g, 25 mmol, 1 eq) and piperidine (3.1 mL, 31
mmol, 1.2 eq) was dissolved in ethyl acetate (125 mL), using sonication
and stirring to assist in dissolving all components. The reaction mixture
was stirred at ambient temperature for 5 h, monitoring the reaction
progress by LC/MS. Upon completion, the solvent was then removed in vacuo
to ˜15 mL, then the product was triturated with hexane (250 mL) to
give an oily residue. Hexane was decanted and the residue was washed
further with hexane (100 mL). The product was dried overnight in vacuo to
provide compound UU (13.5 g) as a yellowish solid. LC-MS [M+H] 451.3
(C23H438N4O5+H, calc: 451.3). Purity >95% (UV/254
nm). Compound UU was used without purification.

[0268] Compound VV (19.2 g, 25 mmol) was suspended in methanol (500 mL)
and filtered off from inorganic salts. A Pd/C (5% wt, 2.4 g) suspension
in water (10 mL) was added, and the reaction mixture was hydrogenated
(Parr apparatus, 80 psi) at ambient temperature for 2 h. Upon reaction
completion, the catalyst was filtered through a pad of Celite® on
sintered glass frit and washed with methanol (2×50 mL). The
filtrate was evaporated in vacuo to give an oily residue. The product was
dried overnight in vacuo to give compound XX (17.3 g) as a pale yellow
oil. LC-MS [M+H] 459.4 (C22H42N4O6+H, calc: 459.3).
Compound XX was used without purification. Purity >95% (UV/254 nm).

In Vitro Trypsin Conversion of Prodrug Compound PC-5 to Hydromorphone and
Inhibition by Trypsin Inhibitor

[0272] This Example demonstrates trypsin conversion of Compound PC-5 to
hydromorphone. Compound PC-5 was exposed to trypsin as described.
Specifically, the reaction included 0.761 mM Compound PC-5.2HCl in the
presence of 0.02 to 0.0228 mg/ml trypsin, 17.5 to 22.5 mM calcium
chloride, Tris pH 8 at 40 to 172 mM, and 0.25% DMSO. A 5-minute
37° C. pre-incubation of the reaction mixture without prodrug was
conducted and then the prodrug was added to initiate the incubation. The
reaction was conducted at 37° C. for 24 hr. Samples were collected
at specified time points, transferred into 0.5% formic acid in
acetonitrile to stop trypsin activity and stored at less than -70°
C. until analysis by LC-MS/MS.

[0273] Table 1 indicates the results of exposure of Compound PC-5 to
trypsin. The results are expressed as half-life of prodrug when exposed
to trypsin (i.e., Prodrug trypsin half-life) in hours and rate of
formation of HM per unit of trypsin.

[0274] The results in Table 1 indicate that trypsin can mediate release
hydromorphone from the Compound PC-5.

[0275] Saline solutions of Compound PC-5 (which can be prepared as
described in Example 9) were dosed as indicated in Table 2A and Table 2B
via oral gavage into jugular vein-cannulated male Sprague Dawley rats (4
per group) that had been fasted for 16-18 hr prior to oral dosing. At
specified time points, blood samples were drawn, harvested for plasma via
centrifugation at 5,400 rpm at 4° C. for 5 min, and 100
microliters (A) plasma transferred from each sample into a fresh tube
containing 2 μl of 50% formic acid. The tubes were vortexed for 5-10
seconds, immediately placed in dry ice and then stored in a -80°
C. freezer until analysis by HPLC/MS.

[0276] Table 2A, Table 2B, FIG. 4A and FIG. 4B provide hydromorphone
exposure results for rats administered different doses of Compound PC-5.
Results in Table 2A and Table 2B are reported, for each group of 4 rats,
as (a) maximum plasma concentration (Cmax) of hydromorphone (HM)
(average±standard deviation), (b) time after administration of
Compound PC-5 to reach maximum hydromorphone concentration (Tmax)
(average±standard deviation) and (c) area under the curve (AUC) from 0
to 24 hr (average±standard deviation) for all doses except for the 1.5
mg/kg Compound PC-5 dose where the AUC was calculated from 0 to 8 hr.

[0277]FIG. 4A and FIG. 4B compared mean plasma concentrations over time
of hydromorphone release following PO administration of increasing doses
of Compound PC-5 for the studies reported in Table 2A and Table 2B,
respectively.

[0279] Saline solutions of Compound PC-5 were dosed at 6 mg/kg with
increasing co-doses of Compound 109 (nafamostat mesylate, Catalog No.
3081, Tocris Bioscience or nafamostat mesylate, Waterstone
WS38665/CAS82956-11-4) as indicated in Table 3 via oral gavage into
jugular vein-cannulated male Sprague Dawley rats (4 per group) that had
been fasted for 16-18 hr prior to oral dosing. At specified time points,
blood samples were drawn, harvested for plasma via centrifugation at
5,400 rpm at 4° C. for 5 min, and 100 microliters (μl) plasma
transferred from each sample into a fresh tube containing 2 μl of 50%
formic acid. The tubes were vortexed for 5-10 seconds, immediately placed
in dry ice and then stored in -80° C. freezer until analysis by
HPLC/MS.

[0280] Table 3 and FIG. 5 provide hydromorphone exposure results for rats
administered with Compound PC-5 and increasing doses of trypsin
inhibitor. Results in Table 3 are reported, for each group of 4 rats, as
(a) maximum plasma concentration (Cmax) of hydromorphone (HM)
(average±standard deviation) and (b) time after administration of
Compound PC-5, to reach maximum hydromorphone concentration
(Tmax)(average±standard deviation) and (c) area under the curve (AUC).

[0285]FIG. 6A and FIG. 6B compare mean plasma concentrations over time of
hydromorphone release following PO administration of a single dose unit
and of multiple dose units of a composition comprising prodrug Compound
PC-5 and trypsin inhibitor Compound 109.

[0286] The results in Table 4A, Table 4B, FIG. 6A and FIG. 6B indicate
that administration of multiple dose units (as exemplified by 2, 3 and 10
dose units of the 109-to-PC-5 (2.2-to 1) dose unit) results in a plasma
hydromorphone concentration-time PK profile that was not dose
proportional to the plasma hydromorphone concentration-time PK profile of
the single dose unit. In addition, the PK profile of the multiple dose
units was modified compared to the PK profile of the equivalent dosage of
prodrug in the absence of trypsin inhibitor.

Example 13

Pharmacokinetics of Hydromorphone Prodrug Following IV Administration to
Rats

[0287] This Example compares the plasma concentrations of prodrug and
hydromorphone in rats following intravenous (IV) administration of
Compound PC-5.

[0288] Compound PC-5 was dissolved in saline and injected into the tail
vein of 4 jugular vein-cannulated male Sprague Dawley rats at a dose of 2
mg/kg. At specified time points, blood samples were drawn, harvested for
plasma via centrifugation at 5,400 rpm at 4° C. for 5 min, and 100
microliters (A) plasma transferred from each sample into a fresh tube
containing 2 μl of 50% formic acid. The tubes were vortexed for 5-10
seconds, immediately placed in dry ice and then stored in -80° C.
freezer until analysis by high performance liquid chromatography/mass
spectrometry (HPLC/MS).

[0291] Table 5 and FIG. 7 demonstrate that the plasma concentration of
hydromorphone in rats administered Compound PC-5 IV is only 0.01% of the
plasma concentration of Compound PC-5, indicating that IV administration
of Compound PC-5 does not lead to significant release of hydromorphone.

Example 14

In Vivo Tolerability of Compound PC-5 in Rats

[0292] This Example demonstrates that Compound PC-5 was tolerated when
administered intravenously to rats.

[0293] Male naive Sprague-Dawley rats, 4 per dose, were used in the study.
Rats were weighed, and then placed under a heat lamp for 15-20 minutes to
dilate the lateral tail veins. Dose volumes are based on the body weights
(1 ml/kg); dosing was as indicated in Table 6. Before dosing, rats were
placed in Broome restrainers and the drug was introduced into one of the
tail veins using a syringe and needle. After dosing, the timer was set
and rats were observed for clinical signs. Blood samples were collected 5
minutes post-dose via the saphenous vein. The rats were observed up to 24
hours.

[0294] The results in Table 6 indicated that rats can tolerate a dose of
9.7 μmol/kg and recover to normal activity within 1.5 hours.

[0295] This Example demonstrates the stability of Compound PC-5 to a
variety of readily available household chemicals and enzyme preparations.

[0296] Compound PC-5 was exposed at room temperature (RT) or 80° C.
for either 1 or 24 hours (hr) to the following household chemicals: vodka
(40% alcohol), baking soda (saturated sodium bicarbonate solution, pH 9),
WINDEX® with Ammonia-D (pH11) and vinegar (5% acetic acid). Compound
PC-5 was also exposed to the following enzyme-containing compositions at
RT for 1 or 24 hr: GNC® Super Digestive (2 capsules of GNC Super
Digestive Enzymes dissolved in 5 ml of water), tenderizer (Adolf's meat
tenderizer, primarily papain, dissolved in water to a concentration of
0.123 g/ml to approximate the concentration of a marinade given on the
bottle label), and subtilisn (8 tablets of ULTRAZYME® contact lens
cleaner (Advanced Medical Optics) dissolved in 4 ml water). Samples were
incubated as described. Aliquots were removed at 1 hr and 24 hr and
stabilized by transferring each to a solution of 50% or 100% phosphoric
acid to achieve a final pH of less than or equal to pH 4. The stabilized
aliquots were then diluted 4- to 6-fold with water, vortex-mixed and
applied to HPLC.

[0297]FIG. 8 demonstrates the release of hydromorphone when Compound PC-5
was exposed to the various household chemicals and enzyme-containing
compositions described above. The percentage of Compound PC-5 remaining
after exposure is indicated by the solid black bars and percentage
conversion of Compound PC-5 to hydromorphone is indicated by the lightly
shaded bars with a black outline. These results indicate that exposure of
Compound PC-5 to these various conditions leads to substantially less
than 10% conversion to hydromorphone.

[0298] While the present invention has been described with reference to
the specific embodiments thereof, it should be understood by those
skilled in the art that various changes may be made and equivalents may
be substituted without departing from the true spirit and scope of the
invention. In addition, many modifications may be made to adapt a
particular situation, material, composition of matter, process, process
step or steps, to the objective, spirit and scope of the present
invention. All such modifications are intended to be within the scope of
the claims appended hereto.

Patent applications by Craig O. Husfeld, San Mateo, CA US

Patent applications by Julie D. Seroogy, San Carlos, CA US

Patent applications by Thomas E. Jenkins, Half Moon Bay, CA US

Patent applications by SIGNATURE THERAPEUTICS, INC.

Patent applications in class IN VIVO DIAGNOSIS OR IN VIVO TESTING

Patent applications in all subclasses IN VIVO DIAGNOSIS OR IN VIVO TESTING